After 5 days of differentiation, myotubes were treated with MLN4924 compound or DMSO for one day then incubated with recombinant rat agrin overnight at 37°C in 5% CO2. Treated cells with recombinant rat agrin were fixed in PFA 4% for 10 minutes. Quantification of AChR expressed at the myotubes surface was determined by incubating fixed cells with FITC-conjugated alpha-bungarotoxin (Molecular Probes, ThermoFisher) for one hour at room temperature. Confocal microscopy was performed using a Zeiss Axioplan 2 upright confocal microscope (Carl Zeiss Inc., Thornwood, NY). Area, number and size of AChR were assessed using ImageJ (1.48v) software [91 ].
Axioplan 2 upright confocal microscope
Manufactured by Zeiss
The Zeiss Axioplan 2 is an upright confocal microscope designed for high-resolution imaging. It features a confocal scanning system that allows for optical sectioning of samples, enabling the capture of detailed, three-dimensional images. The microscope is equipped with a range of objective lenses, providing versatility in magnification and resolution.
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2 protocols using axioplan 2 upright confocal microscope
1
Quantifying AChR Surface Expression in Myotubes
2
Cryosectioning and Immunofluorescence of Embryos
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Fixed embryos were embedded in OCT Tissue-Tek (Thermo Fisher Scientific, Waltham, MA), and sectioned (8 μm) using a Leica CM 3050S cryostat (Leica Microsystems, Bannockburn, IL). Sections were blocked in a solution containing 2% horse serum, 0.2% Triton X-100, and 5% bovine serum albumin in phosphate-buffered saline, followed by incubation with primary antibody overnight at 4°C. The following primary antibodies were used: Cypher24 (link) (1:300), CD31 (1:300; BD Biosciences), sarcomeric alpha(α)-actinin (1:300; Sigma-Aldrich), myomesin (1:300; Santa Cruz Biotechnology), cleaved caspase 3 (1:300; Cell Signaling Technology), and phospho-histone H3 S10 (1:300; Millipore). Subsequently, sections were incubated with fluorescently labeled secondary antibodies. Confocal microscopy was performed using a Zeiss Axioplan 2 upright confocal microscope (Carl Zeiss Inc, Thornwood, NY).
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